Our overall goal is to elucidate biochemical and biophysical aspects of nitric oxide (NO) in sickle cell blood. The use of NO, which is produced naturally, is being considered as a treatment for sickle cell disease. It may benefit patients in its ability to act as a vasodllator, to decrease red blood cell sickling, decrease sickle cell adherence, and to improve oxygen transport. However, the potential of NO to benefit or harm patients cannot be properly assessed without a thorough understanding of its effects in blood, primarily through its interaction with hemoglobin. Much of nitric oxide biology in sickle cell blood, and even in normal blood, is not understood and previous studies on this subject have sometimes reported contradictory results. We will focus on the effects of NO in whole blood or, at least, under conditions that mimic those in vivo as much as possible. We will compare our results from sickle blood to those from studies we conduct on normal blood, on preparations of isolated normal and sickle red blood cells, and on purified hemoglobins prepared under various conditions. We will use a variety of spectroscopic and other biophysical tools, some of which will be developed as part of this project. These include microscopy, ektacytometry, ultracentrifugation, stopped and quench-flow mixing, laser photolysis and diffraction, chemiluminescence, electron spin resonance, nuclear magnetic resonance, and absorption spectroscopy using an integrating sphere detector. We will use these tools to assess nitric oxide's effects on both the kinetics and equilibrium state of red blood cell sickling. We will also study how NO affects the mechanism and kinetics of red blood cell unsickling due to sickle cell polymer melting. We will study the effects of NO on the oxygen affinity of sickle and normal hemoglobin in whole blood and examine the effects of NO on the kinetics of oxygen binding and dissociation. In addition, we will examine how NO effects membrane damage in sickle red blood cells and study the resulting consequences as they relate to red blood cell deformability. Finally, we will study the primary reactions of NO with deoxy and oxy HbS using static and time resolved spectroscopies under physiological conditions. Our studies will help us understand important issues related to NO's role in sickle cell disease and in the general population.